Seasonal Fluctuations of Sex Steroid Hormones in Indian Major Carp Catla Catla in Khouzestan, Iran

Copyright: © 2014 Sehafii HH, et al.. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Seasonal Fluctuations of Sex Steroid Hormones in Indian Major Carp Catla Catla in Khouzestan, Iran Homayoun Hosseinzadeh Sehafii1, Mozhgan khodadadi2 and Shima Ayati Behbahani3* 1Country Fisheries Research Organization, Tehran, Iran 2Aquaculture Department, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran 3Aquaculture Department, Khouzestan Science and Research Branch, Islamic Azad University, Ahvaz, Iran


Introduction
Reproduction is an important factor ensuring the continuation of a species by recruitment of the next generation; and Successful reproduction in seasonally breeding vertebrates depends on temporal variations in gametogenesis, reproductive behaviors, and steroid hormone production [1].
In generally, Reproduction in teleosts, as in other vertebrates, is under hormonal regulation by the brain-pituitary-gonadal axis (BPG axis). The main factors involved in the regulation of reproductive function are pituitary gonadotropins and gonadal steroids. Hormones are regulators which affect cells metamorphism. Hormones are not beginner of reaction and many physiological actions undergo without them. Sex steroids have long been recognized as key hormones regulating sexual differentiation, physiological aspects of reproduction and the development of primary and secondary sexual characteristics [2].
A major estrogen, 17β-estradiol (E2), controls pivotal physiological events in female reproductive cycles in all vertebrates studied to date. The association of changes in gonadal development with plasma levels of sex steroids has proven to be a valuable tool for understanding the endocrine control of reproduction in teleosts. In female teleosts, estradiol-reported that plasma17β-estradiol (E2) levels increased during the vitellogenic stage, but decreased during the maturational stage [3]; while in male teleosts, both 11-ketotestosterone  and testosterone (T) may regulate reproductive morphology and behavior [4]. In many if not all teleosts, progestogens can induce oocyte maturation [5]. The endocrine control cannot continue without appropriate environmental cues required stimulating reproduction [6]. Many external or environmental cues can have profound effects on circulating hormone levels. For example, water temperature, photoperiod, lunar and tidal cycles, and food availability are known to influence spawning behavior, gametogenesis, and circulating steroid levels in fishes [7,8]. Sexual maturity and gonadal development is associated with increased circulating levels of gonadotropins and plasma sex steroids [9].
The major carp Catla (Catla catla) (Cyprinidae, Cypriniformes) is a popular freshwater edible fish and an extensively cultured species in India. This species has been investigated to study its taxonomy, ecology, abundance, feeding and growth parameters, but to date, reproductive biology is quite neglected in many areas [10]. Recently, the exploitation of several valuable cyprinid species for commercial purposes has made their investigation particularly relevant [11]. Catla is a species of choice for the study of temporal organization of reproduction under natural and experimental conditions for understanding environmental and/or endocrine mechanisms that control the annual reproductive cycle [12].
The aim of this study was to investigate the seasonal fluctuations of plasma sex steroids in male (testosterone), and female (testosterone, 17β -estradiol and progesterone) Catla (Catla catla) in Khouzestan.

Abstract
Catla (Catla catla) is one of the most important species of cyprinids in terms of economic and nutritional value that was considered as aquaculture candidate in Khozaestan province (Iran). In this study, 40 female and male Catla broodstocks (imported from India), were studied during year (summer 2011 to spring 2012). Seasonally, blood sampling was collected with heparinated syringe followed by catching and anesthesia. Levels of steroid hormones (testosterone, 17β-estradiol and progesterone) were measured by RIA. Results showed significant differences in all sex steroids during seasonal sampling (P<0.05). High plasma testosterone levels were measured in both sexes (female: 0.13 ± 0.03 ng/ml and male: 0.16 ± 0.04 ng/ml) in the course of the sexual maturation at the end of winter. Highest female plasma levels of 17 β-estradiol (128.50 ± 26.60 ng/ml) was measured in fall. Progesterone also significantly increased at winter (1.32 ± 0.22 ng/ml) in female broodstocks (P<0.05). All broodstocks were ripe and ready to breeding in winter. Water temperature was higth (29.88 ± 1.42) in summer which declined to (14.63 ± 0.47) in winter. According to the results,

Fish supply
The study was conducted between summer 2011 and spring 2012. Totally 40 Catla Broodstocks (5 female and 5 male) were captured from earthen ponds in each season. Fishes were imported from India at 2010 and transported to South Iran Aquaculture Research Center (31° 40' N, 48° 79' E), Ahvaz (Khouzestan Province, Iran). Fish were fed with concentrates fishes (Byza Fars Company) two a day.

Blood sampling
Five samples of both male and female broodstocks were captured seasonally. Fishes were recaptured in each season according to tagged fishes (ABC elastomer Tag, CIST, Canada). The blood samples were taken from caudal vein via heparinized syringe. Plasma was extracted using centrifuge (10 min. at 3000×g) and stored at -20°C until hormone assay [20]. Testosterone (for male and females), 17β-estradiol and progesterone (for females) were measured with radioimmunoassay political approach (RIA) by the commercial Kit of Finland Immune Tech (I 125 ) and gamma counter [21].

Hormone assays
RIA is a quantitative immunoassay technique used to detect the level of protein or antigen in a sample by measuring the diameter of the ring of precipitin formed by the complex of the protein and the antiserum. To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gammaradioactive isotopes of iodine attached to tyrosine [22]. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum from containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen (IgM) in the serum to be derived [23].

Water temperature
Pond water temperature was monthly measured by thermometer. Average water temperature was calculated for each season according to 3 depth data collection.

Statistical analysis
Normality of data was tested by Kolmogorof-Smirnof test. All data were expressed as means ± S.E.M.
The SPSS 15 software was used for statistical analyses and Excel 11 for graphs. Differences between groups were evaluated by analysis of variance (one way ANOVA). The significant differences were determined at (P<0.05).

Testosterone (T)
In the present study level of testosterone hormone in female in spring, summer, autumn and winter seasons were 0.1 ± 0.08 (ng/ ml), 0.04 ± 0.02 (ng/ml), 0.1 ± 0.07 (ng/ml) and 0.13 ± 0.03 (ng/ml) respectively, that higher level belong to winter season .There was significant difference in level of testosterone among spring and fall with summer season (P<0.05). Moreover, there was significant difference in plasma testosterone levels between all seasons with winter season (P<0.05). Lower level of testosterone was observed in summer season ( Figure 1). According to the results, level of testosterone hormone in male in spring, summer, autumn and winter seasons were 0.05 ± 0.02 (ng/ml), 0.04 ± 0.02 (ng/ml), 0 .04 ± 0.05 (ng/ml) and 0.16 ± 0.04 (ng/ml) that higher level belong to winter season and there was significant difference in level of testosterone among winter season with spring, summer and fall (P<0.05). Also, lower this levels of testosterone in male, was observed in summer season ( Figure 1).

Progesterone (P)
In the case of progesterone hormone, higher levels were seen winter season and there was significant difference between winter season with other seasons (P<0.05). Levels of progesterone hormone in spring, summer, autumn and winter seasons were 0.38 ± 0.18 (ng/ml), 0.51 ± 0.4 (ng/ml), 0.46 ± 0.13 (ng/ml) and 1.32 ± 0.22 (ng/ml). This hormone was measured only in female (Figure 3).

Temperature
Average water temperature is measured at different seasons, were obtained as follows (Figure 4)

Discussion
Seasonal changes in the concentrations of circulating sex hormones and their importance for reproduction has been reported for several species of teleosts. Studies have shown that annual fluctuations of hormones related to reproductive, feeding and growth cycles in fishes [15]. Annual rhythm of hormones closely related to factors such as temperature, environment, species of fish, length of day and gonadal sex steroids [2].
Reproduction in fish is under hormonal regulation by the hypothalamus-pituitary-gonadal axis. The brain is stimulated by environmental cues like flooding, water temperature, feeding, rainfall and photoperiod to release gonadotropin releasing hormone [24]. In the pituitary, Gonadotropin Releasing Hormone (GnRH) stimulates the synthesis of Gonadotropin (GtH). GtH-I (or FSH), involved in the initial stages of gametogenesis (vitellogenesis and spermatogenesis), and GtH-II (or LH), which sets FOM, spermiogenesis and spermiation [25]. GtH-I and GtH-II have distinct temporal expression and release profile in teleosts. GtH-I is released over the entire vitellogenesis, whereas GtH-II remains low during vitellogenesis and exhibit a sharp peak before ovulation [26].
The present study was undertaken to investigate changes of serum sex steroids (E2, T and P) and in both sexes Catla catla during the seasonally reproductive cycle. Seasonal changes in circulating plasma levels of sex steroid hormones during the reproductive cycle are described for a variety of teleost species [27,28].
Estradiol is known to be secreted by the cells of the ovarian follicles that promote the development and maintenance of the female sexual characteristics. In humans this hormone (together with other hormones) is responsible for controlling the female sexual cycle. In a variety of species, the level of serum E2 begins to increase in accordance with the appearance of active vitellogenic oocytes, and reaches the highest levels in the tertiary yolk stage oocyte in the ovary, and sharply declines in fish with postvitellogenic and atretic ovaries [29].
According to the results, Plasma E2 levels peak towards the end of vitellogenesis at fall (128.50 ± 26.60 ng/ml) at temperature (20.23 ° 0.69), and they decline rapidly in the maturation stage (88.5 ° 9.50 ng/ml) in winter. Similarly, a peak in 17beta-estradiol plasma concentrations associated with the synthesis of vitellogenic proteins (vitellogenin) has been described in many other teleosts [2]. This result coincided with other studies such as study on Common carp [1], Acipenser Persicus [30], Rutilus firrsi kutum [31], Labeo rohita [32]. In these studies, levels of estradiol had difference during year and its peak was before spawning.
Statistical results showed testosterone at both sex in winter Along with the increase in water temperature in spring (25.47 ° 1.07), was more than other seasons (the stage of sexual maturation); which coincides with after vitellogenesis. Testosterone (T) has been identified in plasma of female teleosts and may be utilized as the major aromatizable androgen [17].
In male teleosts, testosterone is typically elevated during spermatogenesis, and then falls at the onset of spermiation [33]. It appears that a time lapse exists between testosterone production and the male breeding cycle, which is associated with female gonadal maturity. Present result coincided with other researches like study on Rutilus firrsi kutum [3], Capoeta capoeta umbla [34], Acanthopagrus butcheri [35].
Fluctuations in 17β-estradiol paralle led those of androgens, suggesting a close relationship between these steroids. Although the primary role of E2 is to aid in gonadal development, T is involved in other functions, such as positive and negative feedback control of the hypothalamus-pituitary-gonadal axis and migratory behavior in sturgeons [36]. substrate for 17β-Estradiol production in both male and female teleosts [37].
Also the highest level of progesterone level (1.32 ° 0.22 ng/ml) was observed in winter. In female fish, levels of maturation-inducing steroids (usually progesterone) are elevated during final oocyte maturation and ovulation [35]. Progesterone increases in a short period, can indicate a limited role of this hormone on ovarian function and also indirectly involved in the final maturation ovary by hydroxyprogesterone [32]. Levels of progesterone increased with progression of gonadic stage in Huso huso [18].
It is known that the role of sex steroids in controlling the maturation cycle in teleosts especially during spawning times is altered by environmental or hormonal manipulation, and this has both theoretical and practical relevance [38]. Based on the results of this study seem, sex steroid hormones during spawning are changed, the change of seasons and the environment is very effective.
However, due to the great influence of environmental conditions can be said to Khouzestan with suitable climatic conditions such as temperature, the influence of hormonal changes and increase their levels, is considered a good place to culture this fish.